Qualitative and quantitative phytochemical screening of Nerium oleander L. extracts associated with toxicity profile

In this study, phytochemical analysis and toxicity profile of leaf and flower extracts of Nerium oleander L. species collected from Giresun province (Turkey) were investigated. In phytochemical analyzes, the cardiac glycoside, alkaloid, saponin and tannin contents of the extracts were analyzed qualitatively and quantitatively. The physiological effects of extracts were determined by examining root elongation, weight gain and germination rates. Biochemical effects were determined by measuring the levels of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT), which are indicators of oxidative stress. Cytotoxic and genotoxic effects were investigated by mitotic index (MI), micronucleus (MN) and chromosomal abnormality (CA) tests. N. oleander leaf and flower extract applications caused significant decreases in the physiological parameters of Allium bulbs. SOD and CAT activity in root tip cells increased significantly after the application of leaf extract compared to the control group. Similar changes were observed in the application of flower extract, but these increases were found to be at a lower level compared to the increases induced by the leaf extract. An increase in MDA levels and a decrease in GSH levels were observed in groups treated with leaf and flower extracts. These changes show that the extracts cause deterioration in antioxidant/oxidant balance. It was determined that the extracts, which caused a decrease in MI rates and an increase in MN and CAs frequencies, showed the most prominent cytotoxic and genotoxic effects at 250 μg/mL doses. These toxic effects were associated with the phytochemical content of the extracts, and it was thought that cardiac glycosides and alkaloids, whose presence were detected in qualitative and quantitative analyzes, may play an important role in toxicity. Studies investigating the therapeutic properties of plants as well as their toxic effects are insufficient, which leads to the fact that plants exhibiting potential toxicity are not well known. Therefore, this study will lead many studies on the toxicity profile of the phytochemical contents of plants. Therefore, this study will draw attention to the investigation of the toxicity profile and phytochemical contents of plants and will lead to similar studies.

Plants are an important source of a wide variety of secondary metabolites used in the treatment and prevention of diseases 1 . The pharmacological and biological effects of many plants have been elucidated by isolating and characterizing the active ingredients 2 . Today, these active ingredients are also used as pharmaceutical raw materials 3 . The vast majority of drugs are produced from herbal secondary metabolites and compounds derived from these metabolites 4,5 . Secondary metabolites that ensure the survival of the plant have many activities such as antioxidant, antiviral, antimalarial, analgesic, diuretic, anthelmintic, antibacterial, anticancer, antiinflammatory, antifungal, antiallergic and antimutagenic 6 . There are many studies in the literature reporting the protective properties of herbal extracts and compounds isolated from plants against many diseases. The toxic properties of many plant species whose protective properties against many diseases are determined are not investigated and such properties remain in the background. The toxicity of plants especially those consumed as food and used in houses, parks, gardens and various landscaping applications should be investigated. Although there are many literature studies investigating the protective properties of herbal extracts against various damages, studies investigating their toxic properties are not yet at the desired level. In this study, the toxic effects of Nerium oleander L., which has been reported to have a protective effect against cancer in literature, were investigated. www.nature.com/scientificreports/ Test for saponin. To detect the presence of saponin, 5 mL of distilled water was added to 1 mL of extract and vortexed for 10 min. The formation of a foam column that did not disappear with the addition of HCl was evaluated as positive for saponin 27,28 .
Test for tannin. For qualitative tannin analysis, a few drops of lead acetate were added to 1 mL of extract. A large white-brown precipitate formation was considered a positive test for tannin 26 .
Quantitative phytochemical analysis. Quantitative analysis of phytochemical components, whose presence was tested by qualitative analysis, was also carried out.
Cardiac glycoside determination. Cardiac glycoside analysis was determined according to the method reported by Tofighi et al. 29 . 10 g of N. oleander leaf and flower extracts were mixed with 10 mL of Baljet's reagent. After 1 h of incubation, 20 mL of distilled water was added and the absorbance was measured at 495 nm. Securidaside was used as a standard, and the amount of cardiac glycoside was expressed as mg securidaside equivalent (SE)/g.

Tannin determination.
Quantitative tannin content in the extracts was tested according to the method suggested by Mital and Jha 30 . 1 mL of extract was mixed with 0.5 mL of Folin reagent, and then it was saturated with 1 mL of Na 2 CO 3 and 8 mL of distilled water was added to the final mixture. The solution, which was incubated for 30 min, was centrifuged and the supernatant was analyzed at 725 nm. Tannic acid was used as a standard and tannin content was expressed as mg tannic acid equivalent (TAE)/g.
Total saponin determination. The determination of the total saponin content in the extracts was made according to the vanillin-sulfuric acid colorimetric method 31 . The mixture containing 250 µL of vanillin reagent, 50 µL of extract and 2.5 mL of 72% sulfuric acid was mixed and incubated at 60 °C for 10 min. At the end of the incubation, the absorbance of the solution cooled in an ice bath was read at 544 nm. Saponin content was expressed as mg diosgenin equivalent (DE)/g.
Alkaloid determination. Alkaloid determination was made according to the method reported by Selvakumar et al 32 . 2 mL of HCl was added to the extract dissolved in dimethylsulfoxide and the mixture was filtered. An equal volume of bromocresol green and phosphate buffer was added to the solution. The mixture was shaken by adding chloroform. The absorbance of the solutions containing the extract and atropine used as a standard was read at 470 nm. The alkaloid content was expressed as mg atropine equivalent (AE)/g. All tests were repeated in triplicate. Calibration curves of standards were given in Supplementary Fig. S1.  A. cepa bulbs were rinsed with distilled water and their outer scales were removed. Old root remnants were carefully removed to preserve the root primordia. After these preliminary applications, the bulbs were used in toxicity tests. Toxicity was investigated by physiological, biochemical, cytogenetic and anatomical parameters. Seven different groups were formed to investigate the dose-related toxicity of N. oleander leaf and flower extracts (Table 1). EC 50 is defined as the concentration that produces a 50% reduction in root growth. So, inhibitions in Allium root growth were examined to determine the EC 50 value of the extracts. For this purpose, the effects of leaf and flower extract applications in the range of 10-500 μg/mL on root elongation were investigated. EC 50 value was found 300 μg/mL for leaf extract and 425 μg/mL for flower extract. In order to obtain healthy and sufficient root tissue required in toxicity tests, the doses applied in the experimental groups were selected from the values below the EC 50 value of both extracts.
Physiological parameters. The effects of N. oleander leaf and flower extracts on germination were investigated by root length, weight gain, germination percentage and relative injury rate. For this aim, bulbs were placed in glass beakers and germinated in related solutions in the incubator at 22 °C for 72 h. Ten bulbs were used for each group. The solutions of each group were checked daily. At the end of the germination period, the best-developed 10 roots of each bulb were measured and mean root lengths were calculated. Weight gain was calculated by taking the difference between the initial and final weights of the bulbs. Relative injury rate (RIR) and germination percentage (GP) and were calculated using Eqs. (1) and (2), respectively 33 .
Cytotoxic and genotoxic effects. The changes in the mitotic index (MI), micronucleus (MN) and chromosomal abnormalities (CAs) ratios in the Allium test were investigated to determine the cyto-and genotoxic effects of N. oleander leaf and flower extracts. For this purpose, 1 cm long samples were collected from each bulb at the end of the germination period and fixed in Clarke solution. After fixation, the samples washed with 96% ethanol were incubated in 1 N HCl for 17 min at 60 °C for hydrolysis. After the completion of all procedures, the root tips were stained with acetocarmine for 24 h and examined under a research microscope 34 . After germination procedure root slides were prepared and mitotic cells were examined. A total of 10.000 cells were counted for each group and MI percentages were calculated using Eq. (3). Cells in prophase, metaphase, anaphase and telophase were taken as basis in determining the number of dividing cells 35 .
CAs and MN frequencies were investigated in order to determine the genotoxic effects of N. oleander leaf and flower extracts. In the detection of MN and CAs 1000 cells were counted for each group.    MDA and GSH levels. For MDA measurement, 0.5 g sample of each group was homogenized in trichloroacetic acid and centrifuged at 12,000 rpm for 15 min, and MDA analysis was performed in the supernatant. A mixture of 5% thiobarbituric acid and supernatant (1:1) was boiled at 96 °C for 25 min. At the end of the time, the cooled mixture was centrifuged at 10,000 rpm for 5 min and the absorbance of the supernatant was measured at 532 nm 38 . For GSH analysis, the samples belonging to each group were shaken with the same volume of 10% trichloroacetic acid in ice for 15 min and centrifuged at 5000 rpm for 15 min. A mixture containing 0.4 mL of supernatant, 0.8 mL of Tris buffer, and 0.02 mL of DTNB was prepared and the absorbance was read spectrophotometrically at 412 nm after waiting for 5 min 39 .

Anatomical alterations.
To determine the anatomical effects of N. oleander leaf and flower extracts on A.
cepa root tip cells, cross-sections were taken from root tip cells. The sections were stained with methylene blue, examined and visualized with a light microscope. The frequency of anatomical damages was determined by preparing 10 preparations from the samples belonging to each group 40 .
Statistical analysis. Analyzes were performed using the "IBM SPSS Statistics 22" package program and the data obtained were given as mean ± SD (standard deviation). Statistical significance between the means was determined by Duncan's test and One-way ANOVA, and it was considered statistically significant when the p value was < 0.05.

Results and discussion
In this study, the leaf and flower parts of N. oleander samples were collected from Giresun and then the phytochemical analysis and toxic effects were investigated. The phytochemical content was examined by qualitative tests and the toxic effects of the extract were associated with this ingredient.
Qualitative phytochemical analysis. The samples obtained by water and methanol extracts of N. oleander leaf and flowers were used in qualitative-phytochemical analysis. Positive results were obtained in terms of cardiac glycosides, saponins, tannins and alkaloids in N. oleander leaf extract except for tannin in methanol extract (Fig. 2). Cardiac glycosides were detected in the extraction with both solvents and it was determined that the cardiac glycoside content was more intense in the methanol extract. While the presence of saponin was detected in both extracts, the fact that a denser foam formation was observed especially in the extract obtained with water, compared to methanol, indicates that the amount of saponin in this extract is higher. In terms of tannin content, water extract was evaluated as positive and methanol extract as negative. A stronger positivity was determined in the methanol extract of alkaloid content, but a positive result was obtained in terms of the presence of alkaloids in both extracts. In the literature, there are studies in which phytochemical analyzes of various parts of Nerium species are carried out. Bhuvaneshwari et al. 41 performed qualitative analyzes with N. oleander leaf extracts and obtained positive results in terms of terpenoids, alkaloids, cardiac glycosides, saponins and tannins. Rajendra et al. 42 detected the presence of phenolic compounds, alkaloids, tannins, flavonoids and cardiac glycosides in the extracts of N. indicum leaves obtained with benzene and alcohol extraction, and reported negative results in terms of anthroquinone glycoside and carbohydrate. Flower tissues of N. oleander were also analyzed qualitatively by the same phytochemical analyzes and the results are given in Fig. 3. Similar to the leaf extract, cardiac glycosides were detected in both solvents in the flower extract, and the cardiac glycoside content was found to be more intense in the methanol extract. While www.nature.com/scientificreports/ the presence of saponin was not detected in the aqueous extract, very low foam formation was observed in the methanol extract and this result was evaluated as low saponin presence compared to the leaf extract. While negative results were obtained in methanol extract in terms of tannin and alkaloid, positive results were obtained in aqueous extracts. Redha 12 stated that while N. oleander reported the presence of substances such as phenol, tannin, coumarin, alkaloid and sterol in flower tissues, negative results were obtained in terms of saponin and antroquinone glycosides.
Quantitative phytochemical analysis. Quantitative analyzes of the phytochemical ingredients, whose presence were detected by qualitative analysis, were also carried out and the results are given in Fig. 4. Parallel results were obtained with qualitative analyzes, and it was determined that the extract obtained with water gen-  oleander leaf and flower extracts, respectively. The tannin content, which is the major phytochemical in the N. oleander leaf extract, is 456.82 ± 0.33 mgTE/g in the water extract and 77.90 ± 0.11 mgTE/g in the methanol extract. The amount of tannin in N. oleander flower extracts is 46.27% lower than N. oleander leaf extract. A higher amount of saponin was detected in water extracts of N. oleander leaf extract as 22.39 ± 0.09 mgDE/g. Saponin, which was evaluated as negative in N. oleander flower extract water extract in qualitative analyzes, was detected at a rate as low as 2.91 ± 0.08 mgDE/g in quantitative analyzes of N. oleander flower extract. The alkaloid, which is highly found in the methanol extract of N. oleander leaf is more concentrated in the water extract of N. oleander flower. While no alkaloid was detected in the methanol extract of N. oleander flower in qualitative analysis, this substance was detected at low rates in quantitative analysis. In the literature, quantitative phytochemical analyzes have been carried out on Nerium species grown in different ecological conditions. Aslam et al. 43 reported that Nerium indicum collected from Panjgur, Balochistan contains 0.128 g of alkaloids, 0.249 g of saponins and 0.295 g of flavonoids. Bhuvaneshwari et al. 41 reported that N. oleander samples collected from Yercaud (Tamilnadu) contained terpenoids, alkaloids, glycosides, saponins, tannins, and no flavonoids and flobatanine.
Physiological parameters and relative injury rates. The effects of N. oleander leaf and flower applications on germination-related parameters and the relative injury rates are given in Table 2. N. oleander leaf and flower treatments caused a significant decrease in germination parameters in A. cepa bulbs. While 100% germination rate was determined in the bulbs of the control group, the germination percentage was between 72 and 88% in the N. oleander leaf extract applications at 50-250 μg/mL concentrations. The most significant decrease in germination rate was detected in the group treated with 250 μg/mL N. oleander leaf extract, and there was a 1.38-fold decrease in germination rate in this group compared to the control group. N. oleander flower extract application also decreased germination, but this regression remained at lower levels compared to N. oleander leaf extract. In the 250 μg/mL N. oleander flower extract treated group, the germination rate decreased by 1.29 times compared to the control and decreased to 77%. An increase in root length and weight ratio is observed with germination in plants. Germination rates, root elongation and weight gain increased in parallel with each other in control bulbs. The decrease in germination rates caused by N. oleander leaf and flower applications was also detected in root elongation and weight gain, and it was determined that these regressions increased depending on the dose. 250 μg/mL N. oleander leaf and flower applications caused 36.8% and 29.8% reduction in root length compared to the control group, respectively (p < 0.05). 250 μg/mL N. oleander leaf and flower applications caused 48.3% and 40.2% decrease in weight gain compared to the control group, respectively (p < 0.05). When the relative injury rates calculated based on the control group germination percentage were examined, the highest relative injury rate was found as 0.28 in the 250 μg/mL N. oleander leaf extract applied group. The germination-reducing effects of N. oleander leaf and flower applications are closely related to the active ingredients it contains. In this study, the presence of cardiac glycosides in N. oleander leaf and flower extracts was determined by phytochemical analysis. Oleandrin, a cardiac glycoside, whose presence in N. oleander has been determined by many studies, causes disruptions in physiological and biochemical pathways in cells by changing membrane fluidity, increasing intracellular calcium, inducing reactive oxygen species production, oxidative   46 reported that Nerium leaf and flower extracts caused reductions in shoot length, root length and fresh weight of seedlings of monocotyledonous Hordeum vulgare and dicotyledonous Vicia sativa seedlings. Karaaltın et al. 47 reported that Nerium extracts significantly reduced root length in beans and wheat, and this reduction was associated with the allelopathic effect of the extract.
Antioxidant/oxidant dynamics. In order to determine the effects of N. oleander leaf and flower applications on antioxidant/oxidant dynamics, the levels of MDA, GSH, SOD and CAT in root tip cells were examined. SOD and CAT are important endogenous antioxidant enzymes and changes in the activities of these enzymes are closely related to oxidative stress in the cell. N. oleander leaf extract application significantly increased SOD and CAT activity compared to the control group, depending on the dose. The most significant increase was detected in Group IV administered 250 μg/mL N. oleander leaf extract. SOD and CAT activity increased 72.6% and 53.4%, respectively, in Group IV compared to the control group. This shows that the application of N. oleander leaf extract induces the SOD enzyme at a higher rate than the CAT enzyme. Similar increases in enzyme activities were observed in the application of N. oleander flower extract, but these increases were found to be at a lower level compared to the effects of N. oleander leaf extract. Especially, 50 μg/mL and 100 μg/mL N. oleander flower extract applications did not cause a statistically significant (p > 0.05) increase in CAT activity, while 250 μg/mL dose caused a 24.6% increase compared to the control group (p < 0.05). N. oleander flower application caused more significant changes in SOD activity compared to CAT enzyme. While 50 μg/mL and 100 μg/mL N. oleander flower extract administrations provided a similar increase in SOD activity, 250 μg/mL N. oleander flower extract increased SOD activity by 59% compared to the control group (Fig. 5). When all the results were evaluated together, it was determined that N. oleander leaf application significantly induced and increased SOD and CAT activity at a dose of 250 μg/mL. While the application of N. oleander flower extract did not cause a dominant change in enzyme activities, especially at doses of 50 μg/mL and 100 μg/mL, 250 μg/mL dose significantly induced SOD and CAT activities. www.nature.com/scientificreports/ SOD and CAT activities are induced in the presence of oxidative stress and increased enzyme activities after N. oleander leaf and flower applications indicate the formation of oxidative stress in the cell. As a result of biochemical reactions such as respiration and photosynthesis and many metabolic activities in plants, free radicals such as hydroxyl radical, superoxide anion and hydrogen peroxide are formed. These radicals cause oxidative stress in the cell. The antioxidant defense system neutralizes this stress and cellular metabolism continues without interruption. As a result of various agents or stress in cells, exogenous factors increase the production of reactive oxygen species and the activities of antioxidant enzymes are also induced. SOD is an important antioxidant enzyme induced in the presence of oxidative stress and is involved in the dismutation of superoxide radicals 48,49 . The increase observed in SOD activity as a result of the application of N. oleander leaf and flower in this study indicates an increase in oxidative stress. An increase in SOD activity causes an increase in hydrogen peroxide levels in the cell, which induces CAT activity. CAT activity is very important for the survival of plants exposed to stress factors 50,51 . The increases in SOD and CAT activities indicate that N. oleander leaf and flower applications cause oxidative stress in A. cepa root tip cells.
In the N. oleander leaf and flower applied groups, dose-dependent increases were also observed in the MDA levels, with the most significant increase detected in the 250 μg/mL dose of both applications. 250 μg/mL N. oleander leaf and flower applications increased MDA levels in root tip cells by 180.5% and 66.2%, respectively, compared to the control group. Along with the increase in MDA levels, there was a decrease in GSH levels, which is an endogenous and powerful antioxidant. 250 μg/mL N. oleander leaf and flower applications decreased GSH levels in root tip cells by 50.9% and 41.8%, respectively, compared to the control group (Fig. 6). The decrease in GSH levels and increase in MDA levels show that N. oleander leaf and flower applications cause deterioration in antioxidant-oxidant dynamics. MDA is produced at low levels in cells under normal conditions and is used in different pathways such as signal transduction and gene expression. Increases in MDA production are observed in cells under stress conditions and in the presence of oxidative stress. Increases observed in MDA level in a cell indicate oxidative stress and lipid peroxidation that develops accordingly 52 . The fact that N. oleander leaf and flower applications increase the MDA level indicates that oxidative stress is induced. Along with the increase in MDA, a decrease in GSH levels in root tip cells was also determined. GSH is a powerful antioxidant molecule found in various organelles in plants and scavenging free radicals. The decrease in GSH levels indicates that oxidative stress in the cell increases and the reduced-GSH is rapidly oxidized 53 (Table 3). Nuclear bud, vagrant chromosome, bridge, unequal distribution of chromatin, sticky chromosome, fragment and vacuolated nucleus are common CAs observed in both applications (Figs. 8, 9). The frequency of CAs increased with the increase of the extract dose, and the highest frequencies were found in Group IV and Group VII, where 250 μg/mL N. oleander leaf and flower extracts were administered, respectively. N. oleander leaf application caused the highest frequency of sticky chromosome formations among CAs, and N. oleander flower application caused the highest frequency of nuclear bud formation. Chromosomal condensations, DNA depolymerization, and dissolved nucleoproteins induce stickiness in chromosomes. It is also reported in literature studies that sticky chromosome formations cause lethal effects in cells 63,64 . The formation of highly sticky chromosomes as a result of N. oleander leaf administration also supports the reduction in MI rates, and the reduction in cell proliferation is mainly due to irreversible irregularities in mitosis, including stickness. Nuclear bud, which was detected with the highest rate among abnormalities in N. oleander flower application, is closely related to MN formations. High MN frequencies observed as a result of N. oleander leaf and flower applications confirm the nuclear bud and MN relationship. Both nuclear buds and MN are of an aneuploidogenic origin. Nuclear buds separate from the nucleus in the later stages of division to form an MN, and in some cases the buds re-integrate into the nucleus. Nuclear buds also cause chromatin bridges or different chromosomal rearrangements observed in abnormal anaphases. Nuclear buds observed in both extract applications also induce other CAs. Many active ingredients detected in N. oleander leaf and flower extracts may cause CAs by exhibiting genotoxic effects. Cardiac glycosides detected in Nerium flower and leaf extracts have an important place in the genotoxic effect. Cardiac glycosides are potent inhibitors of   (Fig. 10). In the  www.nature.com/scientificreports/ 250 μg/mL N. oleander leaf applied group, epidermis cell damage, flattened cell nucleus, cortex cell damage and thickening of the cortex cell wall were very severe; giant nucleus, binuclear cell, and indistinct conduction tissue were found to be among the low-level damages (Table 4). Anatomical changes in N. oleander flower applied groups occurred at a lower level compared to N. oleander leaf (Fig. 11). Giant nucleus and binuclear cell damage were not detected in the N. oleander flower administration groups. In the group administered 250 μg/mL N. oleander flower, epidermis cell damage, flattened cell nucleus, cortex cell damage and thickening of the cortex cell wall were observed at a moderate frequency, while indistinct vascular tissue was observed at a low frequency (Table 4). These changes in the anatomical structure are defense reactions developed against external agents and aimed at protecting the internal tissues. Thickening of the cortex cell wall occurs as a result of the accumulation of substances such as lignin, cellulose, suberin, and cutin. In this way, the thickening and durability of the cortex cell wall are increased, limiting the entry of various agents, and preventing the access of toxic compounds to the central cylinder and spreading to other tissues 36,68 . Increased expression of lignin-related genes has been reported in plants contaminated with harmful chemicals and pathogens 69,70 . Root cells can increase the number of epidermis cells to prevent exogenous agents from entering the cell. This increase causes deformations and epidermis cell damage by increasing the pressure of the cells on each other. Another anatomical change observed in the study is the flattening of the cell nucleus. The flattened nucleus arises as a result of changes in the physiological, biochemical and DNA integrity of the cell nucleus. The cell nucleus is typically spherical or elliptical in nature, but shape change can occur in response to physical or environmental changes 71,72 . All these changes observed in the anatomical structure of the groups treated with N. oleander leaf and flowers emerge as a result of cumulative effects. There are similar studies in the literature reporting that Nerium extracts cause changes in plant anatomy. Mojarad et al. 33 reported that Nerium extracts significantly increased the root diameter and number of veins in monocotyledonous and dicotyledonous plants compared to the control group.

Conclusion
Many parts of plants can have toxic effects due to the secondary metabolites they contain. Therefore, the potential toxicity of plants commonly used in homes, gardens or landscapes poses a significant risk. It is of great importance to investigate the potential toxicities of plants as well as their protective properties. In the literature, mostly the protective properties of herbal extracts were investigated, and studies on their toxic effects were insufficient. Plant phytochemicals have an important role in the formation of toxicity. Therefore, growing a plant of the same species in different ecological conditions may cause changes in its toxicity profile. In this respect, the fact that the plant diversity in the world is very wide and the phytochemical content of the same plant changes according to the ecological conditions makes the toxicity studies of herbal extracts insufficient. In this study, the toxicity profile and qualitative/quantitative phytochemical analyzes of the flower and leaf parts of the N. oleander collected from Giresun (Turkiye) were investigated. Toxicity studies were investigated using A. cepa, a bioindicator organism, and in this way, possible potential effects in eukaryotes and allelopathic effects of extracts were investigated. Flower and leaf extracts caused regressions in Allium germination and development, and these regressions were thought to be related to oxidative stress in the plant. The fact that extract applications cause changes in antioxidant enzyme levels, increase in MDA levels and decrease in GSH levels confirms oxidative stress. The extracts, which exhibited genotoxic effect by causing higher CAs formation, also showed cytotoxic effects by reducing MI rates and increasing MN frequencies. The toxic effects of leaf and flower extracts can be associated with the active ingredients they contain, and as a result of phytochemical analysis, it was determined that the leaf and flower extracts contained cardiac glycosides, saponins, tannins and alkaloids. Toxicity of both flower and leaf extract may occur as a result of the cumulative action of all active ingredients and may be associated with more than one mechanism. The toxicity of the leaf extract is higher than the flower extract, which can be explained by the higher concentration of the phytochemicals tested in the leaf. The toxic effects of plants used in the garden, home, office and all landscaping applications should not be ignored and reliable environments should be created without using toxic plants if possible. This study, in which the toxic effects of N. oleander leaf and flower extracts were determined, will lead to the investigation of the toxicity profiles of plants due to their phytochemical content.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.